6.5.1 Variável dependente
a) análise da microbioma intestinal.
6.5.2 Variáveis independentes
a) IG: variável contínua (dias) e avaliada pela data da última menstruação confiável ou ultrassom obstétrico precoce, realizado nas primeiras 12 semanas da gestação e confirmada pelo exame clínico neonatal;
b) Peso nascimento: variável numérica contínua, que foi estratificado em faixas de peso;
c) Sexo: masculino/feminino; d) Escore de Apgar;
e) Classificação quanto à IG: em Pequeno (PIG) ou Adequado à idade gestacional (AIG), de acordo com as curvas de crescimento intrauterinas empregadas na rotina da Neonatologia;
f) Dados maternos e obstétricos: tipo de parto, idade materna, presença de bolsa rota superior a 18 horas, infecção urinária materna, corioamninite, descolamento de placenta;
g) Morbidades e intercorrências do recém-nascido: presença de doença da membrana hialina, neutropenia, sepse neonatal precoce e tardia, enterocolite necrosante, convulsões, asfixia neonatal, hemorragia peri-intraventricular, alta ou óbito. Diagnóstico de sepse precoce foi determinado pela presença de hemocultura positiva nas primeiras 72 horas de vida e de sepse tardia, hemocultura positiva após 72 horas de vida, acompanhadas de sinais clínicos de infecção;
h) Tipo de alimentação enteral recebida: leite materno/fórmula láctea/mista (leite materno e fórmula láctea).
Todas essas variáveis foram obtidas pela revisão de prontuários e, longitudinalmente, durante o acompanhamento do recém-nascido na Neonatologia, com o devido registro em ficha protocolo individual (APÊNDICE A).
6.6 SELEÇÃO DA AMOSTRA
Este estudo baseou-se em uma estratégia de amostragem de conveniência com pacientes recrutados na Seção de Neonatologia do Hospital de Clínicas de Porto Alegre (HCPA), no Brasil.
Os grupos foram separados de acordo com o tipo de alimentação: leite materno exclusivo (LME), fórmula láctea exclusiva (FLE), prevalência de leite materno (PLM), prevalência de fórmula láctea (PFL), e misto (fórmula láctea e leite materno (FM)). Os recém- nascidos que foram alimentados com aleitamento materno exclusivo receberam apenas o leite
materno sem adicionar nenhum outro tipo de líquido. Os que foram alimentados com fórmula
láctea exclusiva receberam somente algum tipo de fórmula láctea infantil, sem nenhum tipo de outro alimento. As prevalências de leite materno foram para recém-nascidos que receberam os dois tipos de leite, o materno e algum tipo de fórmula láctea infantil, prevalecendo 70% de leite materno. Já na prevalência de fórmula láctea, receberam os dois tipos de leite, o materno e algum tipo de fórmula láctea infantil, prevalecendo 70% de fórmula láctea infantil. No grupo dos mistos, os recém-nascidos receberam os dois tipos de leite, o materno e algum tipo de fórmula láctea infantil, num percentual de 40-50% de cada tipo.
6.7 CONSIDERAÇÕES ESTATÍSTICAS
Foram digitados os dados no programa Excel e, posteriormente, exportados para o programa SPSS v. 18.0 para análise estatística. Foram descritas as variáveis categóricas por frequências e percentuais. As variáveis quantitativas foram descritas pela média e pelo desvio padrão. Foram comparadas as variáveis categóricas pelo teste de Qui-quadrado e as quantitativas pela Análise de Variância (ANOVA) seguido do teste post-hoc de Tukey.
6.8 CONSIDERAÇÕES ÉTICAS
Atendendo à Resolução 466/2012, que regulamenta a pesquisa com seres humanos, o projeto de pesquisa foi submetido para avaliação quanto aos seus aspectos metodológicos e éticos pela Comissão de Ética e Pesquisa do Hospital de Clínicas de Porto Alegre.
Foi preenchido o Termo de Compromisso para Utilização de Dados, que é fornecido pelo Grupo de Pesquisa e Pós-Graduação do HCPA. Deste modo, foi assegurada a confidencialidade das informações contidas nos bancos que possam identificar os indivíduos.
Também foi dado aos pais o Termo de Consentimento Livre e Esclarecido, para solicitar a autorização para a coleta de fezes e inclusão no estudo (APÊNDICE B).
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8 ARTIGO ORIGINAL
INFLUÊNCIA DA NUTRIÇÃO ENTERAL NA MICROBIOTA INTESTINAL DO RECÉM-NASCIDO PRÉ-TERMO
RESUMO
INTRODUÇÃO: A colonização precoce do intestino neonatal é influenciada pelas práticas de alimentação. A diversidade da microbiota fecal durante a permanência em Unidade de Terapia Intensiva de recém-nascidos (RN) alimentados com leite humano é pouco estudada. OBJETIVO: Determinar as diferenças na microbiota fecal dos prematuros recém-nascidos (PRN), considerando o uso de leite materno exclusivo e de fórmula láctea, ao longo dos primeiros 28 dias de vida. MÉTODO: Foram incluídos 62 PRN com IG ≤ 32 semanas, distribuídos em cinco grupos, conforme regime alimentar: 7 PRN com leite materno exclusivo, 8 PRN com fórmula láctea exclusiva, 16 PRN com alimentação mista > 70% de leite materno próprio, 16 PRN com alimentação mista > 70% de fórmula láctea, e 15 PRN com alimentação mista 50% de leite materno próprio e 50% de fórmula láctea. Critérios de exclusão: infecções congênitas, malformações congênitas e RNs de mães usuárias de drogas. As fezes foram coletadas semanalmente durante os primeiros 28 dias de vida. Todos os espécimes fecais foram dissolvidos em glicerol 1:1 e imediatamente congelados a -80 ºC até extração de DNA microbiano, para amplificação do gene 16 S rRNA e seu sequenciamento. Os dados foram trabalhados por estatística descritiva e analítica. RESULTADOS: Os grupos foram semelhantes em dados perinatais e neonatais. Diferenças significativas na diversidade da comunidade microbiana nos tratamentos foram encontradas (p <0,001), principalmente entre uso de leite materno exclusivo quando comparado à dieta com fórmula láctea exclusiva (33%) e a > 70% de fórmula láctea (37%). A dieta por leite materno exclusivo permitiu maior diversidade microbiana (média de 85 Operational Taxonomic Unit – OTUs), enquanto a > 70% de fórmula láctea a menor diversidade (média de 9 OTUs). Demais grupos apresentaram uma média da diversidade microbiana de 29 OTUs para dieta com fórmula láctea exclusiva, 23 OTUs para > 70% de leite materno próprio e 25 OTUs para 50% de leite materno próprio e 50% fórmula láctea. A proporção média do gênero Escherichia foi sempre maior em tratamentos contendo fórmula láctea do que no tratamento com leite materno exclusivo. CONCLUSÃO: A microbiota fecal no período neonatal de PRN alimentados com leite materno exclusivo possui maior diversidade bacteriana do que os alimentados com fórmula
láctea. Sugerimos que a microbiota fecal determinada pelo uso do leite materno próprio pode ser protetora contra várias morbidades neonatais.
Palavras-chave: Recém-nascido. Microbiota. Alimentação.
ABSTRACT
BACKGROUND: Early colonization of neonatal gut is influenced by feeding practices. Fecal diversity during NICU stay of newborns fed with human milk is poorly studied. OBJECTIVE: To determine the differences in preterm infants´ stool microbiota considering the use of exclusive own´s mothers milk and formula feeding along the first 28 days of life. METHODS: We included newborns with GA ≤ 32 weeks divided in 5 group according the feeding regimen: 7 exclusive own´s mother milk, 8 exclusive preterm formula, 16 mixed feeding with >70% own´s mothers milk, 16 mixed feeding with >70% preterm formula, and 15 mixed 50% own´s mother milk and preterm formula. Exclusion criteria: congenital infections, congenital malformations and newborns of drug addicted mothers. Stools were collected weekly during the first 28 DOL. All specimens were mixed with glycerol 1:1 and immediately frozen at – 80 ºC until microbial DNA extraction, 16 S rRNA amplification and sequencing. RESULTS: All groups were similar in perinatal and neonatal and neonatal data. There were significant differences in microbial community among treatments. Those differences were further confirmed by the permutational multivariate analysis of variance. Approximately 33% and 37% of the variation in distance between microbial communities could be explained by the treatment with maternal milk only compared to diets based exclusively or preferentially in formula, respectively. Alpha diversity measurements indicated significant differences (p <0.001, Krusual-Wallis test) among the diversity of OTUs within treatments. The diet Composed by maternal milk only allowed for greater microbial diversity (average of 85 OTUs). The formula based group presented the smallest diversity (average of 9 OTUs). Those diets based in exclusive formula and preferably maternal milk presented an average of 29 and 23 OTUs respectively; and the diet based in a mixture of formula and maternal milk presented an average of 25 OTUs. The mean proportion of the genus Escherichia was always greater in treatments containing formula than in the treatment with maternal milk only. CONCLUSIONS: Fecal microbiota in the neonatal period of preterm infants fed with exclusive own’s mother milk has increased diversity and a genus composition different from those fed with formula. We suggest that fecal microbiota
determined by use of own’s mother milk may be protective against several neonatal morbidities.
Keywords: Newborn. Microbiota. Feeding.
INTRODUÇÃO
A microbiota intestinal é muito importante para o metabolismo, desenvolvimento e comportamento dos seres humanos.(1,2) Apesar de vários estudos sobre o tema e a sua relação com doenças de alta complexidade(1,3,4), eles restringiram-se apenas à sua enumeração baseada em cultura, perfilhamento genético (16S) e/ou utilização de amostras pequenas, o que deixa claro que os fatores que formam a microbiota intestinal não foram examinados a contento.(1,5,6) Sabe-se que o desenvolvimento da microbiota dos recém-nascidos depende de fatores médicos e dietéticos(1,7), mas não se sabe ainda como eles influenciam a sua